CN112969457A - Combinations of inhibitors of influenza virus replication - Google Patents

Abstract

Provided herein are combinations of compounds that can inhibit influenza virus replication, reduce the amount of influenza virus, and/or treat influenza.

Description

Combinations of inhibitors of influenza virus replication

Technical Field

The present disclosure relates generally to combinations of inhibitors of influenza virus replication, and methods of treating or preventing influenza infection or replication by administering the combinations to a patient in need of treatment thereof.

Background

Influenza spreads worldwide in seasonal epidemics, resulting in hundreds of thousands of deaths per year, with millions of deaths in pandemic years. For example, three influenza pandemics occurred in the 20 th century, and killed tens of millions of people, with each pandemic being caused by the emergence of novel virus strains in humans. Typically, these novel strains are caused by the transmission of existing influenza viruses from other animal species to humans.

Influenza is transmitted from person to person mainly by a large number of virus-carrying droplets produced when an infected person coughs or sneezes; these large droplets may then settle on the mucosal surfaces of the upper respiratory tract of susceptible individuals in the vicinity of (e.g., within about 6 feet of) an infected person. Transmission may also occur by direct or indirect contact with respiratory secretions, such as touching surfaces contaminated with influenza virus and then touching the eyes, nose or mouth. Adults may transmit influenza to others 1 day before symptoms appear to about 5 days after symptoms begin. Young children and people with weak immune systems may remain contagious 10 or more days after the onset of symptoms.

Influenza viruses are RNA viruses of the family orthomyxoviridae, which contains five genera: influenza a virus, influenza B virus, influenza C virus, infectious salmon anemia virus (isavur) and toruloviruses (thogovirus).

Influenza a virus is responsible for seasonal and pandemic influenza. It has one species, influenza a virus, and wild waterfowl is the natural host for many influenza a viruses. Occasionally, the virus spreads to other species and can then cause destructive outbreaks in poultry or cause pandemics of human influenza. Type a viruses are the most virulent human pathogens of the three influenza types and cause the most serious diseases. Influenza a viruses can be subdivided into different serotypes based on antibody responses to these viruses. Among the serotypes identified in humans, ranked by the number of known human pandemic deaths, are: H1N1 (which caused spanish influenza in 1918), H2N2 (which caused asian influenza in 1957), H3N2 (which caused hong kong influenza in 1968), H5N1 (pandemic threat in the influenza season from 2007 to 2008), H7N7 (which is a potential pandemic threat), H1N2 (endemic disease present in humans and pigs), H9N2, H7N2, H7N3 and H10N 7.

Influenza B virus can cause seasonal influenza and has one species, influenza B virus. Influenza B almost exclusively infects humans and is less common than influenza a. The only other animal known to be susceptible to influenza B infection is seal. This type of influenza mutates at a rate 2 to 3 times slower than type a and is therefore less genetically diverse, having only one influenza B serotype. Due to this lack of antigenic diversity, a certain degree of immunity to influenza B is usually obtained in very small time. However, influenza B is sufficiently mutated that sustained immunity is not possible. This reduced rate of antigen change combined with its limited host range (inhibition of cross species antigen transfer) ensures that a pandemic of influenza B does not occur.

Influenza C virus has one species, influenza C virus, which infects humans and pigs and can cause severe disease and localized epidemics. However, influenza C is less prevalent than other types and often appears to cause mild disease in children.

Influenza viruses are very similar in structure in serotype and genus. The influenza virus genome consists of eight single stranded RNAs packed into rod-like structures of different sizes, called ribonucleoprotein complexes (RNPs). Each RNP contains a unique viral RNA, multiple copies of the scaffold nucleoprotein and a heterotrimeric viral polymerase consisting of PA, PB1 and PB2 subunits, which catalyzes transcription and replication of the viral genome. Recent biochemical and structural studies of the influenza polymerase complex provide insight into the mechanistic understanding of cap-abstraction (cap-snatching) and RNA synthesis of influenza polymerases. Briefly, the PB2 end cap binding domain first binds to its 5' end cap to isolate the host pre-mRNA. PA (endonuclease subunit) then cleaves mRNA 10-13 nucleotides downstream of the end cap prior to capture. The PB2 subunit then rotates about 700 to direct the capping primer into the PB1 polymerase active site. The PB1 subunit interacts directly with the PB2 and PA subunits. These subunits contain highly conserved domains in different influenza strains and are of interest as attractive anti-influenza drug targets. In addition to the polymerase complex, the influenza genome encodes its own Neuraminidase (NA), Hemagglutinin (HA), Nucleoprotein (NP), matrix proteins M1 and M2, and nonstructural proteins NS1 and NS 2. NA is the antiviral drug oseltamivir (oseltamivir)

Lanamivir (laninavir)

Peramivir (peramivir) and zanamivir (zanamivir)

The target of (1). These drugs inhibit the enzymatic activity of NA, thereby slowing the release of progeny virus from infected cells.

Influenza incurs direct costs due to lost productivity and associated medical treatment, as well as indirect costs of preventative measures. In the united states, influenza contributes an overall cost of over 100 billion dollars per year, while it is estimated that future pandemics may result in direct and indirect costs of hundreds of billions of dollars. The cost of control is also high. Governments worldwide have spent billions of dollars in preparing and planning for a potential H5N1 avian influenza pandemic, with costs associated with purchasing medications and vaccines and conducting disaster maneuvers and developing strategies for improving border controls.

Current treatment options for influenza include vaccination and chemotherapy or chemoprevention using antiviral drugs. Vaccination against influenza with influenza vaccines is generally recommended for high risk groups, such as children and the elderly, or people with asthma, diabetes or heart disease. However, it is possible that influenza remains after vaccination. The vaccine is reformulated for several specific influenza strains each season, but may not include all strains that effectively infect the population worldwide during that season. Manufacturers spend about six months dispensing and producing millions of doses needed to handle seasonal behavior; occasionally, new or overlooked virus strains become prominent during that period and infect already vaccinated people (e.g. H3N2 fujian flu in the 2003 to 2004 flu season). It may also happen that the infection is just prior to vaccination and is the particular strain against which the vaccine should be prevented, since it takes about two weeks for the vaccine to be effective.

In addition, the effectiveness of these influenza vaccines can vary. Because of the high mutation rate of viruses, certain influenza vaccines typically provide protection for no more than a few years. A vaccine formulated for one year may not be effective the next year because influenza viruses change rapidly over time and different virus strains become dominant.

Due to the absence of RNA correction enzymes, the RNA-dependent RNA polymerase of influenza vRNA produces a nucleotide insertion error approximately every ten thousand nucleotides (which is approximately the length of the influenza vRNA). Thus, almost every newly manufactured influenza virus has a mutant antigen drift. If more than one strain infects a single cell, then separating the genome into eight separate vRNA fragments allows for mixing or reassortment of the vrnas. The resulting rapid changes in viral genetics produce antigen transfer and allow the virus to infect new host species and rapidly overcome protective immunity.

Antiviral drugs may also be used to treat influenza, where NA inhibitors are particularly effective, but the virus may develop resistance to approved NA antiviral drugs. Also, the emergence of multidrug resistant pandemic influenza a virus is well documented. Drug resistant pandemic influenza a becomes a significant public health threat. In addition to resistant influenza a viruses, NA inhibitors are approved for the treatment of early stage influenza infection (within 48 hours of onset of influenza symptoms).

Thus, there remains a need for drugs for treating influenza infection, for example drugs with extended treatment time and/or reduced sensitivity to viral titers.

Disclosure of Invention

The present disclosure relates generally to methods of treating influenza, methods of inhibiting influenza virus replication, methods of reducing the amount of influenza virus, and compounds and compositions useful in these methods.

Provided herein are methods of treating or preventing influenza infection or replication in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of (1)3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid or a pharmaceutically acceptable salt or solvate thereof, and (2) a second influenza therapeutic agent, the second influenza therapeutic agent being selected from the group consisting of: baloxavir, a neuraminidase inhibitor and favipiravir (favipiravir), or a pharmaceutically acceptable salt or solvate thereof.

In addition, the present disclosure provides a combination comprising (1)3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid or a pharmaceutically acceptable salt or solvate thereof, and (2) baroxavir or baroxavir bosch, or a pharmaceutically acceptable salt or solvate thereof; and (3) neuraminidase inhibitors such as oseltamivir, oseltamivir acid, zanamivir, lanamivir, peramivir, or pharmaceutically acceptable salts or solvates thereof.

Further provided are methods of administering a safe and effective amount of a combination as disclosed herein to a biological sample or patient.

Also provided herein are methods of reducing the amount of influenza virus in a biological sample or patient by administering to the biological sample or patient a safe and effective amount of a combination as disclosed herein.

Further provided are methods of treating or preventing influenza a or influenza B infection in a patient comprising administering to the patient a safe and effective amount of a combination as disclosed herein.

Also provided is the use of a combination described herein for inhibiting or reducing the replication of influenza virus in a biological sample or a patient, for reducing the amount of influenza virus in a biological sample or a patient, or for treating influenza in a patient.

Further provided herein is the use of a combination described herein for the manufacture of a medicament for treating influenza in a patient, for reducing the amount of influenza virus in a biological sample or a patient, or for inhibiting replication of influenza virus in a biological sample or a patient.

The combinations disclosed herein may be co-formulated or may be administered to an individual, patient or host separately.

Drawings

Figure 1 is a macsynergy tmii scheme showing the role of compound 1 in combination with baroxavir in analyzing influenza virus a/PR/8/34(H1N1) replication in MDCK cells. Exhibit synergy/antagonism compared to the 95% confidence interval. Throughout this text, compound 1 refers to 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid.

FIG. 2 is a MacSynergy showing the role of Compound 1 in combination with oseltamivir acid in analyzing influenza A/PR/8/34(H1N1) replication in MDCK cells^TMII scheme. Exhibit synergy/antagonism compared to the 95% confidence interval.

FIG. 3 is a MacSynergy demonstrating the role of Compound 1 in combination with Favipiravir in the analysis of influenza A/PR/8/34(H1N1) replication in MDCK cells^TMII scheme. Exhibit synergy/antagonism compared to the 95% confidence interval.

Detailed Description

Disclosed herein are combinations of anti-influenza compounds (antiviral agents), and the use of these combinations in inhibiting influenza virus activity. In some aspects, the present disclosure generally relates to the use of a composition described herein for inhibiting the replication of influenza virus in a biological sample or patient, for reducing the amount of influenza virus in a biological sample or patient (reducing viral titer) and for treating or preventing influenza in a patient. The combinations disclosed herein may be co-formulated or may be administered to an individual, patient or host separately.

The combination comprises in particular 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid or a pharmaceutically acceptable salt or solvate thereof as active ingredient, alternatively referred to herein as "compound 1". This compound is believed to be a PB2 domain inhibitor that binds CAP.

In various embodiments, compound 1 is used in combination with a second anti-viral agent as a prophylactic or therapeutic agent against influenza, e.g., against influenza virus replication or infection. Influenza can be a pandemic or drug resistant pandemic/seasonal influenza virus. In some cases, the influenza is influenza a or influenza B. The second antiviral agent may be a polymerase inhibitor, endonuclease inhibitor or neuraminidase inhibitor or an influenza vaccine. Further discussion regarding the timing of administration of compound 1 and the second antiviral agent is provided below.

In some aspects, a combination is provided comprising 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid or a pharmaceutically acceptable salt or solvate thereof and a second antiviral agent, and in some cases, the second antiviral agent is selected from the group consisting of: barosavir, Barosavirenz, a neuraminidase inhibitor and Favipiravir, or a pharmaceutically acceptable salt or solvate thereof.

When administered separately, compound 1 and the second antiviral agent can be administered simultaneously (e.g., within about 5 to 10 minutes of each other), or 1 or more hours apart (e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 24 hours, 26 hours, 48 hours, or 72 hours). In some cases, compound 1 can be administered prior to the second antiviral agent. In some other cases, compound 1 can be administered after the second antiviral agent. Further discussion regarding the timing of administration of compound 1 and the second antiviral agent is provided below.

In some aspects, a combination is provided comprising a therapeutically effective amount of a)3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid or a pharmaceutically acceptable salt or solvate thereof and b) a therapeutically effective amount of a second antiviral agent, and in some cases, the second antiviral agent is selected from the group consisting of: barosavir, Barosavirenz, a neuraminidase inhibitor and Favipiravir, or a pharmaceutically acceptable salt or solvate thereof.

In other aspects, there is provided the use of a therapeutically effective amount of a combination as disclosed herein for the treatment or prevention of influenza virus infection or replication in a human patient. For example, the influenza virus may be a pandemic or drug resistant pandemic/seasonal influenza virus. In some cases, the influenza virus may be influenza a or influenza B. Other influenza viruses are described below.

In other aspects, there is provided the use of 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid, or a pharmaceutically acceptable salt or solvate thereof, in combination with a second antiviral agent for the manufacture of a medicament for the treatment or prevention of influenza virus infection or replication, and in some cases, the second antiviral agent is selected from the group consisting of: barosavir, Barosavirenz, a neuraminidase inhibitor and Favipiravir, or a pharmaceutically acceptable salt or solvate thereof.

In other aspects, there is provided the use of 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid, or a pharmaceutically acceptable salt or solvate thereof, in combination with a second antiviral agent for inhibiting influenza virus infection or replication, and in some cases, the second antiviral agent is selected from the group consisting of: barosavir, Barosavirenz, a neuraminidase inhibitor and Favipiravir, or a pharmaceutically acceptable salt or solvate thereof.

In other aspects, there is provided a method of treating or preventing influenza virus infection or replication, comprising administering to a human patient having or at risk of influenza virus infection a combination of: a) a therapeutically effective amount of 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid or a pharmaceutically acceptable salt or solvate thereof, and b) a therapeutically effective amount of a second antiviral agent, in some cases, selected from the group consisting of: barosavir, Barosavirenz, a neuraminidase inhibitor and Favipiravir, or a pharmaceutically acceptable salt or solvate thereof.

In other aspects, there is provided a method of treating or preventing influenza virus infection or replication, comprising administering to a human patient having or at risk of influenza infection a dose of about 10 to 1,000mg/kg of 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid or a pharmaceutically acceptable salt or solvate thereof, and a therapeutically effective amount of a second anti-viral agent, in some cases, selected from the group consisting of: barosavir, Barosavirenz, a neuraminidase inhibitor and Favipiravir, or a pharmaceutically acceptable salt or solvate thereof.

In other aspects, there is provided a pharmaceutical composition for treating or preventing influenza virus infection or replication in a patient, comprising 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid or a pharmaceutically acceptable salt or solvate thereof as an active ingredient, wherein the composition is administered in combination with a second antiviral agent, in some cases selected from the group consisting of: barosavir, Barosavirenz, a neuraminidase inhibitor and Favipiravir, or a pharmaceutically acceptable salt or solvate thereof.

In other aspects, there is provided a method of inhibiting the endonuclease activity of an influenza a or influenza B polymerase in a subject, the method comprising contacting the subject with a combination of compound 1 and a second antiviral agent as disclosed herein.

In other aspects, there is provided a method for treating or preventing influenza a or influenza B infection in a host, the method comprising administering to the host a therapeutic amount of compound 1 as disclosed herein in combination with a second antiviral agent.

In other aspects, there is provided a method for reducing the endonuclease activity of an influenza a polymerase or influenza B polymerase in a host, comprising administering to the host a therapeutic amount of compound 1 as disclosed herein in combination with a second antiviral agent.

In other aspects, there is provided a method for reducing influenza virus replication in a host, the method comprising administering to the host a therapeutic amount of compound 1 as disclosed herein in combination with a second antiviral agent.

In some embodiments, methods of using the disclosed combination of compound 1 and a second antiviral agent are provided, further comprising contacting an influenza virus with or administering to a host a therapeutically effective amount of a third antiviral agent. For example, in some embodiments, the method may further comprise administering an influenza vaccine to the host before, after, or simultaneously with the combining. In some cases, the disclosed methods comprise administering compound 1 and an influenza vaccine without administering another antiviral agent (i.e., the vaccine is a second antiviral agent). In some cases, compound 1 is administered concurrently with the influenza vaccine. In some cases, compound 1 is co-formulated with an influenza vaccine.

In other aspects, there is provided a use of compound 1 as disclosed herein in combination with a second antiviral agent for the treatment of an influenza a or influenza B virus infection.

In other aspects, there is provided a use of compound 1 as disclosed herein in combination with a second antiviral agent for the manufacture of a medicament for the treatment of an influenza a or influenza B virus infection.

In other aspects, a combination is provided comprising a)3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid or a pharmaceutically acceptable salt or solvate thereof, b) baroxavir, baroxavir macbecylate or a pharmaceutically acceptable salt or solvate thereof, and c) a neuraminidase inhibitor.

The neuraminidase inhibitor for the methods disclosed herein can be ospitabineWeir, oseltamivir acid, zanamivir

Lanamivir

Or peramivir, or a pharmaceutically acceptable salt or solvate thereof. Endonuclease inhibitors can be used in the methods disclosed herein, and in some cases, endonuclease inhibitors are referred to as "PA" inhibitors. In various embodiments, the endonuclease inhibitor may be baroxavir or baroxavir bosch ester, or a pharmaceutically acceptable salt or solvate thereof. Polymerase inhibitors may be used in the methods disclosed herein, and in some cases, the polymerase inhibitors are referred to as "PB 1" inhibitors. In various embodiments, the polymerase inhibitor may be favipiravir or a pharmaceutically acceptable salt or solvate thereof. Influenza vaccines can be used in the methods disclosed herein.

In various embodiments of aspects disclosed herein, the second antiviral agent is baroxavir, baroxavir macbecylate, or a pharmaceutically acceptable salt or solvate thereof. In other embodiments of aspects disclosed herein, the second antiviral agent is a neuraminidase inhibitor (e.g., more specifically oseltamivir, oseltamivir acid or a pharmaceutically acceptable salt or solvate thereof). In other embodiments of aspects disclosed herein, the second antiviral agent is fapiravir or a pharmaceutically acceptable salt or solvate thereof.

Application method

The combinations described herein can be used to reduce viral titer of a biological sample (e.g., infected cell culture) or viral titer of a human (e.g., pulmonary viral titer of a patient).

As used herein, the terms "influenza virus-mediated condition," "influenza infection," or "influenza" are used interchangeably to mean a disease caused by influenza virus infection.

Influenza is an infectious disease caused by influenza viruses that affects birds and mammals. Influenza viruses are RNA viruses of the family orthomyxoviridae, which contains five genera: influenza a virus, influenza B virus, influenza C virus, infectious salmon anemia virus, and torpedo virus. Influenza a virus genus has one species, influenza a virus, which can be subdivided into different serotypes based on antibody responses to these viruses: H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H7N9, and H10N 7. Influenza B virus has one species, influenza B virus. Influenza B infects humans almost exclusively and is less prevalent than influenza a. Influenza C virus has one species, influenza C virus, which infects humans and pigs and can cause severe disease and local epidemics. However, influenza C viruses are less prevalent than other types and often appear to cause mild disease in children.

In some embodiments, the influenza or influenza virus is associated with an influenza a or B virus. In some embodiments, the influenza or influenza virus is associated with influenza a virus. In some particular embodiments, the influenza a virus is H1N1, H2N2, H3N2, H7N9, or H5N 1. In some embodiments, the disclosed combinations are effective in inhibiting the growth or replication of a pandemic or drug-resistant pandemic/seasonal influenza virus.

In humans, common symptoms of influenza are chills, fever, pharyngitis, muscle aches, severe headaches, coughing, weakness and general malaise. In more severe cases, influenza causes pneumonia, which can be fatal, especially for young children and the elderly. Although it is often confused with the cold, influenza is a much more serious disease and is caused by different virus types. Influenza can cause nausea and vomiting, particularly in children, but these symptoms are more characteristic of unrelated gastroenteritis, which is sometimes referred to as "gastric flu" or "24 hour flu".

Symptoms of influenza can begin quite suddenly one to two days after infection. Typically, the first symptom is chills or intolerance of cold, but fever is equally prevalent early in the infection, with body temperatures ranging from 38 ℃ to 39 ℃ (about 100 ° f to 103 ° f). Many people are so ill that they are bedridden for several days with general pain and discomfort that is more severe in their backs and legs. Symptoms of influenza may include: general pain (especially in joints and throat), extreme cold and fever, weakness, headache, eye irritation tearing, eye redness, skin (especially facial) redness, mouth redness, throat redness and nasal redness, abdominal pain (children with influenza type B). Symptoms of influenza are not specific, but rather overlap with many pathogens ("influenza-like illness"). Often, laboratory data is required to confirm the diagnosis.

The terms "disease," "disorder," and "condition" are used interchangeably herein to refer to an influenza virus-mediated medical or pathological condition.

As used herein, the terms "individual", "host" and "patient" are used interchangeably. The terms "individual", "host" and "patient" refer to an animal (e.g., a bird or mammal such as a chicken, quail or turkey), particularly a mammal, such as a non-primate (e.g., a cow, pig, horse, sheep, rabbit, guinea pig, rat, cat, dog or mouse) and a primate (e.g., a monkey, chimpanzee or human), and more particularly a human. In some embodiments, the subject is a non-human animal, such as a farm animal (e.g., a horse, cow, pig, or sheep), or a pet (e.g., a dog, cat, guinea pig, or rabbit). In a preferred embodiment, the subject is a human.

The term "biological sample" as used herein includes (but is not limited to): a cell culture or extract thereof; a biopsy material obtained from a mammal or an extract thereof; blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof.

As used herein, the term "inhibiting replication of influenza virus" includes both reducing the amount of viral replication (e.g., by at least 10%) and completely inhibiting viral replication (i.e., reducing the amount of viral replication by 100%). In some embodiments, replication of the influenza virus is inhibited by at least 50%, at least 65%, at least 75%, at least 85%, at least 90%, or at least 95%.

Influenza virus replication can be measured by any suitable method known in the art. For example, the influenza virus titer of a biological sample (e.g., an infected cell culture) or the influenza virus titer of a human (e.g., the pulmonary virus titer of a patient) can be measured. More particularly, for cell-based assays, in each case where cells are cultured in vitro, the virus is added to the culture in the presence or absence of a test agent, and the virus-dependent endpoint is assessed after a suitable length of time. For a typical assay, Madin-Darby canine kidney cells (MDCK) and standard tissue cultures appropriate for the influenza strain A/Puerto Rico/8/34 can be used. The first type of cell analysis that can be used depends on the death of the infected target cells, a process known as cytopathic effect (CPE), where viral infection causes depletion of the cell source and eventual lysis of the cells. In the first type of cell assay, a smaller fraction of the cells in the wells of a microtiter plate are infected (typically 1/10 to 1/1000), the virus undergoes several rounds of replication in 48 to 72 hours, and the amount of cell death is then measured using the reduction in cellular ATP content compared to uninfected controls. The second type of cellular analysis that can be used depends on the proliferation of virus-specific RNA molecules in the infected cells, where RNA levels are directly measured using the branched-chain DNA hybridization method (bDNA). In the second type of cell analysis, a lower number of cells are initially infected in the wells of a microtiter plate, allowing the virus to replicate in the infected cells and spread to additional rounds of cells, followed by cell lysis and measurement of viral RNA content. This analysis is usually stopped early after 18 to 36 hours, when all target cells are still viable. Viral RNA is quantified by hybridization to specific oligonucleotide probes immobilized to the wells of the assay tray, followed by amplification of the signal by hybridization to additional probes linked to a reporter enzyme.

As used herein, "viral titer" or "titer" is a measure of viral concentration. Titer testing can employ serial dilutions to obtain approximate quantitative information from analytical procedures that only evaluate positive or negative in nature. Titer corresponds to the highest dilution factor that still produced a positive reading; for example, positive readings in the first 8 consecutive two-fold dilutions were converted to titers of 1: 256. A specific example is virus titer. To determine titre, several dilutions will be prepared, e.g. 10^-1、10^-2、10^-3、…、10^-8. Feeling of stayingThe lowest virus concentration staining cells was the virus titer.

As used herein, the term "treatment" refers to both therapeutic and prophylactic treatment. For example, therapeutic treatment includes causing a reduction or slowing of the progression, severity, and/or duration of an influenza virus-mediated condition, or ameliorating one or more symptoms (particularly, one or more discernible symptoms) of an influenza virus-mediated condition, by administration of one or more therapies (e.g., one or more therapeutic agents, such as a compound or composition described herein). In particular embodiments, the therapeutic treatment includes ameliorating at least one measurable physical parameter of an influenza virus-mediated condition. In other embodiments, the therapeutic treatment includes physically inhibiting the development of an influenza virus-mediated condition, for example, by stabilizing discernible symptoms, physiologically, for example, by stabilizing physical parameters, or both. In other embodiments, the therapeutic treatment comprises reducing or stabilizing an influenza virus-mediated infection. Antiviral drugs can be used in the community setting to treat people already with influenza, to reduce the severity of symptoms and to reduce their number of days of illness.

As used herein, the terms "prevention", "prophylactic use" and "prophylactic treatment" refer to any medical or public health procedure aimed at preventing, rather than treating or curing, a disease. As used herein, the term "preventing" refers to reducing the risk of developing or developing an established condition, or reducing or inhibiting the recurrence of or the condition in an individual who is not ill but who has become or may be near to becoming a sick person. The term "chemoprevention" refers to the prevention of a disorder or disease using a drug, such as a small molecule drug (rather than a vaccine).

Prophylactic uses include use in situations where outbreaks have been detected to prevent infection from being transmitted or spread in locations where many people at high risk of serious influenza complications live in close contact with each other (e.g., hospitals, nursery houses, prisons, elderly nurseries, etc.). It also includes use in people who need protection from influenza but do not obtain protection after vaccination (e.g. due to a weaker immune system), or when the vaccine is not available for it or when it is not available due to side effects. It also includes use within two weeks after vaccination or during any period after vaccination but before the vaccine is effective. Prophylactic use may also include treating a person who is not suffering from influenza or is not considered to have a high risk of complications to reduce the chance of infection with influenza and transmitting it to a person at high risk of being in close contact with him/her (e.g., health care workers, nursing home workers, etc.).

As used herein and in accordance with the United States Centers for Disease Control and Prevention, (US CDC), an influenza "outbreak" is defined as a sharp increase in acute febrile respiratory Disease (AFRI) occurring within a 48 to 72 hour period in people in proximity to each other (e.g., in the same area of a assisted living facility, in the same household, etc.) relative to normal background rates or when any individual in the analyzed population tests positive for influenza.

In some embodiments, the combination is useful as a prophylactic or preventative measure against patients, particularly humans, susceptible to complications caused by influenza virus infection. The combination may be useful in prophylactic methods in situations where it is established that an indication of a case or outbreak is present, to prevent the spread of infection in the community or the rest of the population.

As used herein, "effective amount" refers to an amount sufficient to elicit a desired biological response. In the present disclosure, the desired biological response is to inhibit replication of influenza virus, to reduce the amount of influenza virus, or to reduce or ameliorate the severity, duration, progression, or onset of influenza virus infection, to prevent the acceleration of influenza virus infection, to prevent the recurrence, manifestation, onset, or progression of symptoms associated with influenza virus infection, or to enhance or improve the prophylactic or therapeutic effect of another therapy used against influenza infection. The precise amount of compound administered to an individual will depend on the mode of administration, the type and severity of the infection, and the characteristics of the individual (e.g., general health, age, sex, weight, and tolerance to drugs). One of ordinary skill in the art will be able to determine the appropriate dosage based on these and other factors.

Thus, when 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid is co-administered with other antiviral agents, for example when co-administered with an anti-influenza drug, the effective amount of the second agent will depend on the type of drug used. A safe amount is one that has the least number of side effects or acceptable side effects and severity, as can be readily determined by one of ordinary skill in the art. Suitable dosages for approved agents are known and can be adjusted by the person of ordinary skill in the art depending on the condition of the individual, the type of condition being treated, and the amount of the compound described herein being used. In the case where the amount is not explicitly labeled, a safe and effective amount should be assumed. For example, 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid, or a pharmaceutically acceptable salt or solvate thereof, can be administered to a subject at a dosage range of between about 0.01 and 100mg/kg body weight/day.

As used herein, a "safe and effective amount" of a compound or composition described herein is an effective amount of the compound or composition that does not cause excessive or harmful side effects in a patient.

In general, the dosage regimen will be selected in accordance with a variety of factors including: the condition being treated and the severity of the condition; the activity of the particular compound employed; the particular composition employed; the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration, and rate of excretion of the particular compound employed; the renal and hepatic function of the individual; and the particular compound or salt employed, the duration of treatment; drugs used in combination or concomitantly with the particular compound employed; and similar factors well known in the medical arts. A safe and effective amount of a compound described herein that is required for treating, preventing, inhibiting (in whole or in part), or arresting disease progression can be readily determined by the person of ordinary skill in the art and established.

The dosage of the compounds described herein may range between about 0.01 to about 100mg/kg body weight/day, about 0.01 to about 50mg/kg body weight/day, about 0.1 to about 50mg/kg body weight/day, or about 1 to about 25mg/kg body weight/day. It is understood that the total amount per day may be administered in a single dose, or may be administered in multiple administrations, such as twice a day (e.g., every 12 hours), three times a day (e.g., every 8 hours), or four times a day (e.g., every 6 hours).

For therapeutic treatment, a compound described herein can be administered to a patient within, e.g., 48 hours (or within 40 hours, or less than 2 days, or less than 1.5 days or 24 hours) of the onset of symptoms (e.g., nasal congestion, sore throat, cough, pain, weakness, headache, and cold/night sweats). Therapeutic treatment may be continued for any suitable duration, e.g., 5 days, 7 days, 10 days, 14 days, etc. For prophylactic treatment during a community outbreak, a compound described herein can be administered to a patient, e.g., within 2 days of the onset of symptoms indicative of a case, and can last for any suitable duration, e.g., 7 days, 10 days, 14 days, 20 days, 28 days, 35 days, 42 days, etc.

Combination therapy

The combinations described herein can be administered alone or in further combination with additional suitable therapeutic agents (e.g., a third antiviral agent or vaccine). When using combination therapy, a safe and effective amount may be achieved using a first amount of compound 1, i.e., 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid or a pharmaceutically acceptable salt or solvate thereof, a second antiviral agent, and an amount of one or more third antiviral agents. In some cases, the third antiviral agent is a pyrazine carboxamide antiviral compound, an influenza neuraminidase inhibitor, or an influenza PB1 polymerase domain inhibitor. Other therapeutic combinations may be achieved by additional suitable therapeutic agents (e.g., antiviral agents or vaccines).

A second antiviral agent suitable for use in these combinations is Barosavir (CAS No. 1985605-59-1), which is a prodrug of mebacoxil (CAS No. 1985606-14-1; trade name

) Shionogi Co. (a Japanese medical doctor)Drug companies) are developing for the treatment of influenza a and influenza B.

Another suitable second antiviral agent for these combinations is oseltamivir phosphate (CAS number 204255-11-8; trade name;)

Which is a neuraminidase inhibitor under development by Roche pharmaceutical company as a drug for treating or preventing influenza infection.

Another second antiviral agent suitable for use in these combinations is Favipiravir (CAS number 259793-96-9; T-705; trade name

) A pyrazine carboxamide derivative developed by Toyama Chemical co.ltd. (a japanese medicinal company) for the treatment of RNA viruses, including influenza a and influenza B. The 200mg troche was approved for the treatment of influenza in japan. For adults, Favipiravir may be administered orally in an amount of 10mg to 10,000mg per day. In some embodiments, favipiravir may be administered orally in an amount of 100mg to 4,000mg per day. In some embodiments, favipiravir may be administered orally on day 1 in an amount of 1,600mg twice a day, and on days 2 through 5 in an amount of 600mg twice a day, with a total administration period of five days.

Kitano et al reported that the combination of baroxavir and neuraminidase inhibitors synergistically inhibited influenza a/H1N 1 virus replication in MDCK cells (Open form Infectious Diseases), 4, Issue application — 1, 2017, 10/1, p. S371). Thus, in some embodiments, there is provided a combination comprising 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid and either baroxavir or baroxavir bosch ester and a neuraminidase inhibitor. Neuraminidase inhibitors suitable for use in this combination include, for example, oseltamivir acid, zanamivir, lanamivir, and peramivir, or more specifically oseltamivir phosphate, oseltamivir acid, zanamivir hydrate, lanamivir, and peramivir trihydrate.

In some embodiments of the present disclosure, compound 1, or a pharmaceutically acceptable salt thereof, and the second antiviral agent are each administered in a safe and effective amount (i.e., each in an amount that will be therapeutically effective when administered alone). In some embodiments, compound 1 and the second antiviral agent are each administered in an amount that alone does not provide a therapeutic effect (sub-therapeutic amount). In some embodiments, compound 1 can be administered in a safe and effective amount, while the second antiviral agent is administered in a sub-therapeutic amount. In some embodiments, compound 1 can be administered in sub-therapeutic amounts, while the second antiviral agent is administered in a safe and effective amount.

As used herein, the terms "combination therapy," "combination," and "co-administration" or "co-administration" are used interchangeably to refer to the use of more than one therapy (e.g., one or more prophylactic and/or therapeutic agents). The use of the terms does not limit the order in which therapies (e.g., prophylactic and/or therapeutic agents) are administered to an individual.

Co-administration may encompass administration of the combined first and second amounts of the compound in a substantially simultaneous manner, e.g., in a single pharmaceutical composition, e.g., in a capsule or tablet having a fixed ratio of the first and second amounts, or in a plurality of separate capsules or tablets. Furthermore, these co-administrations may also encompass the use of each compound in combination in a sequential manner, in either order.

In some embodiments, the present disclosure is directed to a combination therapy method for inhibiting influenza virus replication in a biological sample or a patient, or treating or preventing influenza virus infection in a patient, using a compound or pharmaceutical composition of the present disclosure. Accordingly, the pharmaceutical compositions described herein also include those compositions comprising 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid in combination with a second antiviral agent that exhibit anti-influenza virus activity.

Methods of use also include combinations of compound 1 with a second antiviral agent, with other combinations of another antiviral agent, and/or vaccination with an influenza vaccine.

Where co-administration comprises administering a first amount of 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid and a second amount of a second anti-viral agent alone, 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid and the second antiviral agent are administered sufficiently close in time to have the desired therapeutic effect. For example, the time period between each administration can range from minutes to hours and can be selected to produce the desired therapeutic effect by considering the characteristics (e.g., performance, solubility, bioavailability, plasma half-life, and kinetic profile) of each compound. For example, 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid and the second antiviral agent can be administered in any order within about 24 hours of each other, within about 16 hours of each other, within about 8 hours of each other, within about 4 hours of each other, within about 1 hour of each other, or within about 30 minutes of each other.

More particularly, 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid can be administered prior to (e.g., 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), simultaneously with or after (e.g., 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, a first antiviral agent, a second antiviral agent, or a second antiviral agent, After 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks).

The methods of co-administering an amount of 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid and an amount of a second antiviral agent as set forth herein can produce an enhanced or synergistic therapeutic effect, wherein the observed combined effect is greater than the additive effect expected from administering the amount of compound 1 and the amount of the second antiviral agent alone.

As used herein, the term "synergistic" refers to a combination of the present disclosure that is more effective than the additive effects of the component compounds. The synergistic effect of a combination of therapies (e.g., a combination of prophylactic or therapeutic agents) can allow for the use of lower doses of one or more therapies and/or less frequent administration of the therapies to an individual. Being able to utilize lower doses of therapy (e.g., prophylactic or therapeutic agents) and/or less frequently administering the therapy can reduce the toxicity associated with administering the therapy to an individual without reducing the efficacy of the therapy in preventing, treating, or treating a condition. In addition, the synergy may result in improved efficacy of the agent in preventing, treating, or treating the condition. Finally, the synergistic effect of a combination of therapies (e.g., a combination of prophylactic or therapeutic agents) can avoid or reduce adverse or undesirable side effects associated with the use of either therapy alone.

In addition, when using the combination therapies of the present disclosure, the component therapeutic agents can be administered such that the time period between each administration can be longer (e.g., days, weeks, or months).

The presence of synergy can be determined using suitable methods for assessing drug interactions. Suitable methods include, for example, the Sigmoid-Emax equations (Holford, N.H.G., and Scheiner, L.B., "clinical pharmacokinetics (Clin. Pharmacokinet.). 6: 429-453(1981)), Loewe additive equations (Loewe, S, and Muischnek, H.," archive experiment pharmacology and pathology (Arch. exp. Pathol. Pharmacol.). 114: 313-326(1926)) and median effect equations (Chou, TC, and Talalay, P., "Adv. enzyme Regulation research progress (adv. enzyme Regul.). 22: 27-55 (1984)). Each of the above mentioned equations can be applied in conjunction with experimental data to generate a corresponding graph that helps to assess the effect of the drug combination. The corresponding graphs associated with the above mentioned equations are the concentration-effect curve, the isobologram curve and the combined index curve, respectively. MacSynergy^TMII is well-established graphic software suitable for calculating combinatorial indices (Prichard and Shipman, 1990).

Vaccine against influenza

The compounds described herein can be administered prophylactically in conjunction with an anti-influenza vaccine. These vaccines can be administered, for example, by subcutaneous or intranasal administration. Vaccination by subcutaneous injection typically induces IgG antibodies in the serum with neutralizing activity and is extremely effective in preventing the progression of conditions to more severe conditions such as pneumonia and similar diseases. However, IgA is the major prophylactic component in the upper respiratory mucosa as the site of infection. Since IgA is not induced by subcutaneous administration, it may also be advantageous to administer vaccines by the intranasal route.

Definitions and general terms

The compounds described herein are defined herein by their chemical structures and/or chemical names. When a compound is referred to by chemical structure and chemical name, and the chemical structure contradicts the chemical name, the chemical structure determines the identity of the compound.

Pharmaceutically acceptable salts and solvates

The compounds described herein may be present in free form or, where appropriate, in the form of a salt. Those salts which are pharmaceutically acceptable are of particular interest as they are suitable for administration of the compounds which are components of the described combinations for medical purposes. Non-pharmaceutically acceptable salts are useful in manufacturing processes for isolation and purification purposes, and in some instances, for isolating stereoisomeric forms of the compounds described herein or intermediates thereof.

As used herein, the term "pharmaceutically acceptable salt" refers to salts of compounds which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue side effects (such as toxicity, irritation, allergic response, and the like) commensurate with a reasonable benefit/risk ratio.

Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in detail in journal of medical Sciences (j. pharmaceutical Sciences), 1977,66,1-19, by s.m. berge et al, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds described herein include those derived from suitable inorganic and organic acids and bases. These salts can be prepared in situ during the final isolation and purification of the compounds.

In the case where the compounds described herein contain a base or sufficiently basic bioisostere, acid addition salts can be prepared by 1) reacting the purified compound in its free base form with a suitable organic or inorganic acid and 2) isolating the salt thus formed. In practice, acid addition salts may be in a more suitable form for use, and the use of such salts is equivalent to the use of the free base form.

Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or by using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, formates, fumarates, glucoheptonates, glycerophosphates, glycolates, gluconates, glycolates, hemisulfates, heptanoates, hexanoates, hydrochlorides, hydrobromides, hydroiodides, 2-hydroxy-ethanesulfonates, lactobionates, lactates, laurylsulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoate, and the like, Pectate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate and the like.

In the case where the compounds described herein contain a carboxylic acid group or a sufficiently acidic bioisostere, the base addition salts can be prepared by 1) reacting the purified compound in its acid form with an appropriate baseA synthetic organic or inorganic base and 2) isolating the salt thus formed. In practice, the base addition salts may be used in a more suitable form, and the use of the salt form itself corresponds to the use of the free acid form. Salts derived from suitable bases include alkali metal (e.g., sodium, lithium, and potassium) salts, alkaline earth metal (e.g., magnesium and calcium) salts, ammonium salts, and N⁺(C_1-4Alkyl radical)₄And (3) salt. The present disclosure also contemplates the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water-or oil-soluble or dispersible products can be obtained by such quaternization.

Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include sodium, potassium, calcium, barium, zinc, magnesium and aluminum. Sodium and potassium salts are generally preferred. Other pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium and amine cations formed using counter ions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates and aryl sulfonates. Suitable inorganic base addition salts are prepared from metal bases including sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide, and the like. Suitable amine base addition salts are prepared from amines which are frequently used in medicinal chemistry because of their low toxicity and acceptability for medical use. Ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N' -benzhydrylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris (hydroxymethyl) -aminomethane, tetramethylammonium hydroxide, triethylamine, benzhydrylamine, diphenylhydroxymethylamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, dicyclohexylamine, and the like.

Other acids and bases, when not pharmaceutically acceptable per se, may be used to prepare salts useful as intermediates in obtaining the compounds described herein and pharmaceutically acceptable acid or base addition salts thereof.

The components of such combinations may include mixtures/combinations of different pharmaceutically acceptable salts and mixtures/combinations of the compound in free form and a pharmaceutically acceptable salt.

The components of the combination may be present in the form of solvates. The term "solvate" refers to a molecular complex of a compound (including salts thereof) with one or more solvent molecules. These solvent molecules are those commonly used in pharmaceutical technology that are known to be harmless to the recipient, such as water, ethanol, dimethyl sulfoxide, acetone, and other common organic solvents. The term "hydrate" refers to a molecular complex comprising a compound and water.

Pharmaceutical composition

The compounds described herein can be formulated into pharmaceutical compositions further comprising a pharmaceutically acceptable carrier, diluent, adjuvant, or vehicle. In some embodiments, the disclosure relates to a pharmaceutical composition comprising a compound described herein and a pharmaceutically acceptable carrier, diluent, adjuvant, or vehicle. In some embodiments, the disclosure includes a pharmaceutical composition comprising a safe and effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, adjuvant, or vehicle. Pharmaceutically acceptable carriers include, for example, pharmaceutical diluents, excipients or carriers appropriately selected with respect to the intended form of administration and in accordance with conventional pharmaceutical practice.

An "effective amount" includes both a "therapeutically effective amount" and a "prophylactically effective amount". The term "therapeutically effective amount" refers to an amount effective in treating and/or alleviating influenza virus infection in a patient. The term "prophylactically effective amount" refers to an amount effective in preventing and/or substantially reducing the chance or size of an outbreak of an influenza virus infection.

Pharmaceutically acceptable carriers can contain inert ingredients that do not unduly inhibit the biological activity of the compound. A pharmaceutically acceptable carrier should be biocompatible, e.g., non-toxic, non-inflammatory, non-immunogenic, or free of other undesirable reactions or side effects following administration to an individual. Standard pharmaceutical compounding techniques may be employed.

Pharmaceutically acceptable carriers, adjuvants, or vehicles as used herein include any solvent, diluent, or other liquid vehicle, dispersing or suspending aid, surfactant, isotonic agent, thickening or emulsifying agent, preservative, solid binder, lubricant, and the like as appropriate for the particular dosage form desired. Remington's Pharmaceutical Sciences, 16 th edition, e.w. martin (Mack Publishing co., Easton, Pa), 1980, discloses various carriers used in formulating pharmaceutically acceptable compositions and known techniques for their preparation. Unless any conventional carrier medium is incompatible with the compounds described herein, e.g., by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component of a pharmaceutically acceptable composition, its use is contemplated to be within the scope of the present disclosure.

As used herein, the term "side effect" encompasses an undesirable and undesirable effect of a therapy (e.g., prophylactic or therapeutic agent). Side effects are always undesirable, but undesirable effects are not necessarily adverse. An adverse effect of a therapy (e.g., prophylactic or therapeutic agent) can be harmful or uncomfortable or at risk. Side effects include, but are not limited to, fever, chills, lethargy, gastrointestinal toxicity (including gastrointestinal ulcers and erosions), nausea, vomiting, neurotoxicity, nephrotoxicity (including conditions such as papillary necrosis and chronic interstitial nephritis), hepatotoxicity (including elevated serum liver enzyme content), bone marrow toxicity (including leukopenia, myelosuppression, thrombocytopenia, and anemia), xerostomia, metallic taste, prolonged pregnancy, weakness, somnolence, pain (including muscle disorders, bone pain, and headache), hair loss, fatigue, dizziness, extravertebral symptoms, akathisia, cardiovascular disorders, and sexual dysfunction.

Some examples of materials that can serve as pharmaceutically acceptable carriers include (but are not limited to): an ion exchanger; alumina; aluminum stearate; lecithin; serum proteins (e.g., human serum albumin); buffer substances (e.g. twin 80, phosphate, glycine, sorbic acid or potassium sorbate); partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (e.g. protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride or zinc salts); colloidal silicon dioxide; magnesium trisilicate; polyvinylpyrrolidone; a polyacrylate; a wax; a polyethylene-polypropylene oxide block copolymer; methyl cellulose; hydroxypropyl methylcellulose; lanolin; sugars such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; a powder of tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; no pyrogen water; isotonic saline; ringer's solution; ethanol; and a phosphate buffer solution; and other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

Formulations for pulmonary delivery

In some embodiments, the pharmaceutical compositions described herein are suitable for administration directly to the lower respiratory tract (e.g., the lungs) via inhalation. Compositions for administration by inhalation may be in the form of inhalable powder compositions or liquid or powder sprays and may be administered in standard form using powder inhalation devices or aerosol dispensing devices. Such devices are well known. For administration by inhalation, powdered formulations typically comprise the active compound together with an inert solid powdered diluent (e.g., lactose or starch). The inhalable dry powder compositions may be presented in capsules and cartridges of gelatin or similar material or blisters of laminated aluminum foil for use in an inhaler or insufflator. Each capsule or cartridge may typically contain, for example, from about 10mg to about 100g of each active compound. Alternatively, the compositions described herein may be presented without excipients.

The inhalable compositions may be packaged for unit-dose or multi-dose delivery. For example, the composition may be packaged for multiple dose delivery in a manner similar to that described below: GB2242134, U.S. patent nos. 6,632,666, 5,860,419, 5,873,360 and 5,590,645 (all illustrate "Diskus" devices); or GB2i78965, GB2129691, GB2169265, U.S. patent nos. 4,778,054, 4,811,731 and 5,035,237 (which illustrate "Diskhaler" devices); or EP 69715 ("Turbuhaler" device) or GB 2064336 and us 4,353,656 ("Rotahaler" device).

Spray compositions for topical delivery to the lungs by inhalation may be formulated in the form of an aqueous solution or suspension or aerosol delivered from pressurized packs, such as Metered Dose Inhalers (MDI), using suitable liquefied propellants, including hydrofluoroalkanes, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and especially 1,1,1, 2-tetrafluoroethane, 1,1,1,2,3,3, 3-heptafluoro-n-propane and mixtures thereof. Aerosol compositions suitable for inhalation may be presented in the form of a suspension or solution.

Drugs administered by inhalation generally have a controlled particle size. The most preferred particle size for inhalation into the bronchial system is typically from about 1 μm to about 10 μm, and in some embodiments, from about 2 μm to about 5 μm. Particles having a particle size greater than about 20 μm are generally too large to reach the smaller respiratory tract upon inhalation. To achieve these particle sizes, the particles of the active ingredient may be subjected to a particle size reduction process, for example, micron-sized. The desired particle size fraction may be separated by air classification or sieving. Preferably, the particles will crystallize.

Intranasal sprays may be formulated with aqueous or non-aqueous vehicles with the addition of agents such as thickening agents, buffer salts or acids or bases to adjust pH, isotonic adjusting agents or antioxidants.

Solutions for inhalation by nebulization may be formulated using aqueous vehicles with the addition of agents such as acids or bases, buffer salts, isotonic adjusting agents or antibacterial agents. It may be sterilized by filtration or heating in an autoclave, or presented as a non-sterile product. The nebulizer supplies the aerosol in the form of a mist generated from the aqueous formulation solution.

In some embodiments, the pharmaceutical compositions described herein may be formulated with supplemental active ingredients.

In some embodiments, the pharmaceutical compositions described herein are administered from a dry powder inhaler.

In other embodiments, the pharmaceutical compositions described herein are optionally incorporated by an aerosol dispensing device, for example

Inhalation chamber of the inhalation chamber.

The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Prevention of the action of microorganisms in the compositions described herein can be achieved by the addition of antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferred to include isotonic agents, for example sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

In some embodiments, the pharmaceutical compositions described herein can be within a matrix that controls the release of the composition. In some embodiments, the matrix may comprise: lipids, polyvinyl alcohols, polyvinyl acetates, polycaprolactones, poly (glycolic acid), poly (lactic acid), polycaprolactones, polylactic acids, polyanhydrides, polylactide-co-glycolides, polyamino acids, polyethylene oxides, acrylic acid-capped polyethylene oxides, polyamides, polyethylene, polyacrylonitrile, polyphosphazenes, poly (orthoesters), Sucrose Acetate Isobutyrate (SAIB), and combinations thereof, as well as other polymers disclosed, for example, in U.S. patent nos. 6,667,371, 6,613,355, 6,596,296, 6,413,536, 5,968,543, 4,079,038, 4,093,709, 4,131,648, 4,138,344, 4,180,646, 4,304,767, 4,946,931, each of which is expressly incorporated herein by reference in its entirety. In these embodiments, the matrix provides sustained release of the drug.

The pharmaceutically acceptable carrier and/or diluent may also include any solvent, dispersion medium, coating, antibacterial and/or antifungal agent, isotonic and absorption delaying agent, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in pharmaceutical compositions is contemplated.

The pharmaceutical compositions described herein may be formulated for administration according to conventional techniques. See, for example, Remington, The Science and Practice of medicine (20 th edition, 2000). For example, the intranasal pharmaceutical compositions of the present disclosure may be formulated as aerosols (this term includes both liquid and dry powder aerosols). As known to those having ordinary skill in the art, aerosols of liquid particles may be generated by any suitable means, such as using a pressure-driven aerosol atomizer or an ultrasonic atomizer. See, for example, U.S. Pat. No. 4,501,729. Aerosols of solid particles (e.g., lyophilized, freeze-dried, etc.) may likewise be generated by techniques known in the medical arts using any solid particulate medicament aerosol generator. As another example, the pharmaceutical compositions of the present disclosure can be formulated in an on-demand soluble form that provides a lyophilized portion of the pharmaceutical composition and a dissolved solution portion of the pharmaceutical composition.

In some embodiments of the present disclosure, the pharmaceutical composition is in the form of an aqueous suspension, which may be prepared from a solution or suspension. For solutions or suspensions, the dosage form may consist of micelles of lipophilic substances, liposomes (phospholipid vesicles/membranes) and/or fatty acids (e.g., palmitic acid). In particular embodiments, the pharmaceutical composition is a solution or suspension capable of dissolving in a fluid secreted by the epithelial cell mucosa of the tissue to which the pharmaceutical composition is administered, administered and/or delivered, which may advantageously enhance absorption.

The pharmaceutical composition may be aqueous, non-aqueous or a combination of aqueous and non-aqueous.

Suitable aqueous solutions include (but are not limited to): hydrogels, aqueous suspensions, aqueous microsphere dispersions, aqueous liposome dispersions, aqueous micelles of liposomes, aqueous microemulsions and any combination of the foregoing or any other aqueous solution that is soluble in the fluids secreted by the nasal mucosa. Exemplary non-aqueous solutions include (but are not limited to): non-aqueous gels, non-aqueous suspensions, non-aqueous microsphere dispersions, non-aqueous liposome dispersions, non-aqueous emulsions, non-aqueous microemulsions and any combination of the foregoing or any other non-aqueous solution that is soluble or miscible in the fluid secreted by the mucosa.

Examples of powder formulations include (but are not limited to): pure powder mixtures, micronized powders, freeze-dried powders, lyophilized powders, powder microspheres, coated powder microspheres, liposome dispersions, and any combination of the foregoing. The powder microspheres may be formed from a variety of polysaccharides and celluloses, including, but not limited to, starch, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, polyvinyl alcohol alginate, acacia, polyglucose, and any combination thereof.

In particular embodiments, an inhalable composition is one that is at least partially or even substantially (e.g., at least 80%, 90%, 95%, or more) soluble in fluids secreted by the mucosa for absorption. The composition may be formulated with carriers and/or other substances that facilitate dissolution of the agent into the secretions, including, but not limited to, fatty acids (e.g., palmitic acid), gangliosides (e.g., GM-1), phospholipids (e.g., phosphatidylserine), and emulsifiers (e.g., polysorbate 80).

Those skilled in the art will appreciate that for intranasal administration or delivery, nasal secretions can alter the pH of the administered dose as the pH range in the nasal cavity can be as broad as 5 to 8, since the amount of pharmaceutical composition administered is typically small. Such changes may affect the concentration of unionized drug available for absorption. Thus, in representative embodiments, the pharmaceutical composition further comprises a buffer to maintain or adjust the pH in situ. Typical buffers include, but are not limited to, ascorbate, acetate, citrate, gluten, carbonate, and phosphate buffers.

In some embodiments, the pH of the pharmaceutical composition is selected such that the internal environment of the mucosal tissue after administration is neutral or acidic, which (1) can provide the active compound in an unionized form for absorption, (2) inhibits the growth of pathogenic bacteria that are more likely to be present in an alkaline environment, and (3) reduces the likelihood of irritation of the mucosa.

For liquid and powder sprays or aerosols, the pharmaceutical compositions may be formulated to have any suitable and desired particle size or droplet size. In illustrative embodiments, the majority and/or average particle size of the particles or droplets ranges from equal to or greater than about 1, 2.5, 5, 10, 15, or 20 microns and/or equal to or less than about 25, 30, 40, 45, 50, 60, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, or 425 microns (including all combinations of the foregoing). Representative examples of suitable ranges for the majority and/or average particle size or droplet size include, but are not limited to, about 5 to 100 microns, about 10 to 60 microns, about 175 to 325 microns, and about 220 to 300 microns, which facilitate deposition of a safe and effective amount of the active compound, for example, in the nasal cavity (e.g., in the upper third of the nasal cavity, the upper nasal passage, the olfactory region, and/or the sinus region leading to the olfactory nerve pathway). Generally, particles or droplets smaller than about 5 microns will deposit in the trachea or even the lungs, while particles or droplets of about 50 microns or larger generally cannot reach the nasal cavity and deposit on the anterior portion of the nose.

International patent publication WO 2005/023335 describes particles and droplets having a diameter size suitable for practicing representative embodiments of the present disclosure. In particular embodiments, the particles or droplets have an average diameter of about 5 to 30 microns, about 10 to 20 microns, about 10 to 17 microns, about 10 to 15 microns, about 12 to 17 microns, about 10 to 15 microns, or about 10 to 12 microns. The particles may "generally" have an average diameter or size as described herein, i.e., at least about 50%, 60%, 70%, 80%, 90%, or 95% or more of the particles have a specified diameter or size range.

The pharmaceutical compositions described herein may be delivered in the form of a nebulized or nebulized liquid having a droplet size as described above.

According to particular embodiments involving intranasal delivery methods, it may be desirable to extend the residence time of the pharmaceutical composition in the nasal cavity (e.g., in the upper third of the nasal cavity, the upper nasal passage, the olfactory region, and/or the sinus region), e.g., to enhance absorption. Thus, the pharmaceutical composition may optionally be formulated with a bioadhesive polymer, a gum (e.g., xanthan gum), a polyglucose (e.g., a highly purified cationic polysaccharide), pectin (or any carbohydrate that thickens or emulsifies like a gel when applied to the nasal mucosa), microspheres (e.g., starch, albumin, polydextrose, cyclodextrin), gelatin, liposomes, carbomers, polyvinyl alcohol, alginates, acacia, polyglucose, and/or cellulose (e.g., methyl or propyl cellulose; hydroxy or carboxy cellulose; carboxymethyl or hydroxypropyl cellulose), which are agents that increase the residence time in the nasal cavity. As another approach, increasing the viscosity of the formulation may also provide a means of prolonging contact of the agent with the nasal epithelium. The pharmaceutical composition may be formulated as a nasal emulsion, ointment or gel, which provides the advantage of topical administration due to its viscosity.

Moist and highly vascularized membranes may promote rapid absorption. Thus, the pharmaceutical composition may optionally comprise a humectant (especially in the case of gel-type compositions) to ensure sufficient intranasal moisture content. Examples of suitable humectants include, but are not limited to, glycerol (glycerin/glycerol), mineral oil, vegetable oil, membrane regulators, soothing agents, and/or sugar alcohols (e.g., xylitol, sorbitol; and/or mannitol). The concentration of the humectant in the pharmaceutical composition will vary depending on the agent and formulation selected.

The pharmaceutical composition may also optionally include absorption enhancers, such as agents that inhibit enzyme activity, reduce viscosity or elasticity of mucus, reduce mucociliary clearance effects, open tight junctions, and/or solubilize the active compound. Chemical enhancers are known in the art and include chelating agents (e.g., EDTA), fatty acids, cholates, surfactants, and/or preservatives. Enhancers for permeation may be particularly useful when formulating compounds that exhibit poor membrane permeability, lack lipophilicity, and/or are degraded by aminopeptidases. The concentration of the absorption enhancer in the pharmaceutical composition will vary depending on the agent and formulation selected.

Preservatives may optionally be added to the pharmaceutical composition in order to prolong shelf life. Suitable preservatives include, but are not limited to, benzyl alcohol, parabens, thimerosal, chlorobutanol, and benzalkonium chloride, and combinations of the foregoing. The concentration of the preservative will vary depending on the preservative used, the compound formulated, the formulation and the like. In representative embodiments, the preservative is present in an amount of about 2% by weight or less.

The pharmaceutical compositions described herein may optionally contain an odorant, for example as described in EP 0504263B 1, to provide a sensation of the odor in order to facilitate inhalation of the composition, thereby facilitating delivery to the olfactory region and/or triggering transmission by olfactory neurons.

As another option, the composition may include a flavoring agent, for example, to enhance taste and/or individual acceptability of the composition.

Porous particles for pulmonary administration

In some embodiments, the particles are porous such that they have a suitable density to avoid deposition in the back of the throat when administered by an inhaler. The combination of relatively large particle size and relatively low density avoids phagocytosis in the lung, provides for properly targeted delivery, avoids systemic delivery of components and provides for high concentrations of components within the lung.

Representative methods for making and for delivering these particles are described in, for example, U.S. patent No. 7,384,649, U.S. patent No. 7,182,961, U.S. patent No. 7,146,978, U.S. patent No. 7,048,908, U.S. patent No. 6,956,021, U.S. patent No. 6,766,799, and U.S. patent No. 6,732,732.

Other patents disclosing these particles include U.S. patent No. 7,279,182, U.S. patent No. 7,252,840, U.S. patent No. 7,032,593, U.S. patent No. 7,008,644, U.S. patent No. 6,848,197, and U.S. patent No. 6,749,835.

U.S. patent No. 7,678,364 discloses a method for delivering particles to the pulmonary system comprising: administering to the respiratory tract of a patient in need of treatment, prevention, or diagnosis a safe and effective amount of a dry powder comprising: a) a polyvalent metal cation complexed with a therapeutic, prophylactic or diagnostic agent; b) a pharmaceutically acceptable carrier; and c) a polyvalent metal cation-containing component, wherein the dry powder is spray-dried and has a total polyvalent metal cation amount of about 10% w/w or more, about 0.4g/cm, based on the total weight of the medicament³Or lower tap density, a median geometric diameter of from about 5 microns to about 30 microns, and an aerodynamic diameter of from about 1 to about 5 microns.

The amount of a compound described herein or salt thereof present in the particles can range from about 0.1 wt% to about 95 wt%, although in some cases, can even be as high as 100%. For example, from about 1 to about 50 weight percent, such as from about 5 to about 30 weight percent. Particles in which the drug is distributed throughout the particle may be preferred.

In some embodiments, the particles comprise a surfactant other than the phospholipids described above. As used herein, the term "surfactant" refers to any agent that preferentially absorbs to the interface between two immiscible phases (e.g., the interface between water and an organic polymer solution, a water/air interface, or an organic solvent/air interface). Surfactants generally have a hydrophilic portion and a lipophilic portion such that upon absorption into the particle they tend to bring the portions to an external environment that does not attract particles like the coating, thus reducing particle aggregation. Surfactants may also facilitate the absorption of therapeutic or diagnostic agents and increase the bioavailability of the agents.

Suitable surfactants that may be used in the manufacture of the particles described herein include (but are not limited to): cetyl alcohol; fatty alcohols, such as polyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; surface-active fatty acids, such as palmitic acid or oleic acid; glycocholate; surfactin (surfactin); poloxamers (poloxamers); sorbitan fatty acid esters, e.g. sorbitanPear sugar alcohol trioleate

80; and tyloxapol (tyloxapol).

The surfactant may be present in the particles in an amount in the range of about 0 to about 5 weight percent. Preferably, it may be present in the particles in an amount in the range of about 0.1 to about 1.0 wt%, for example 1.0 wt%.

Having a density of less than about 0.4g/cm³Particles of a tap density of at least about 5 μm, a median diameter of about 1 μm to about 5 μm, or an aerodynamic diameter of about 1 μm to about 3 μm are more capable of avoiding inertial and gravitational deposition in the oropharyngeal region and are targeted to the respiratory tract or deep lung. The use of larger, more porous particles is advantageous because it enables more efficient aerosolization than smaller, more dense particles, such as those currently used in inhalation therapy.

Liposome delivery

The compositions described herein are advantageously delivered to the lung to provide the compound at the site of actual or potential influenza infection. This can be achieved by pulmonary delivery through a metered dose inhaler or other pulmonary delivery device, and also by passing the particles into the microvascular bed surrounding the alveoli in the lung.

Nanocarriers (e.g., liposomes) comprising smaller unilamellar vesicles exhibit several advantages over other conventional methods for drug delivery to the lung, including prolonged drug release and cell-specific targeted drug delivery. Nanoscale drug carriers can also be advantageous for delivery of poorly water soluble drugs, and some of the compounds described herein are poorly water soluble. Additional advantages include its ability to provide controlled release, protection from metabolism and degradation, reduced drug toxicity and targeting ability.

Liposomes (preferably unilamellar vesicles) have a size of less than 200nm as measured by dynamic light scattering, and are preferably characterized by being composed of chemically pure synthetic phospholipids, most preferably having side chains of at least 16 carbons in length, and containing one or more of the compounds described herein or a pharmaceutically acceptable salt thereof sufficient to preferentially deliver (i.e., target) an amount of its compound to the microvascular bed surrounding the alveoli. The vesicle diameter can be measured, for example, by dynamic light scattering using a helium-neon 100mW NEC gas laser and a Malvern K7027 correlator, ideally producing at least two or three measurements at a time for each sizing.

The expression "chemically pure phospholipids" is intended to define phospholipids substantially free of harmful decontaminating moieties and impurities causing aggregation of the Smaller Unilamellar Vesicles (SUV) formed therefrom and having a purity of more than 97%. Preferably, the liposomes have a diameter of predominantly about 50 to about 160nm, are substantially neutral in charge, and incorporate phospholipids having side chains of 16 to 18 carbon atoms in length. More preferably, the liposomes are prepared from Distearoylphosphatidylcholine (DSPC) and include cholesterol (most preferably in an amount of 10% to 50% of the total lipids) as a vesicle stabilizer.

It may also be advantageous for the liposomes to have a melting point above body temperature (i.e., greater than 37 ℃). For this reason, it may be advantageous to use pure phospholipids, preferably phospholipids which are saturated and have a carbon chain length of at least 16 carbons, preferably between 16 and 18 carbons. Distearoylphosphatidylcholine (DSPC) is a preferred phospholipid. Cholesterol helps stabilize the liposomes and is preferably added in an amount sufficient to provide liposome stability. Most preferably, the liposome further comprises a pegylated phospholipid, such as dspeg. The methods involve introducing into the bloodstream of a patient an amount of liposomes that are less than 200nm in size (preferably unilamellar vesicles) and are preferably characterized by comprising a chemically pure synthetic phospholipid, most preferably having a side chain of at least 16 carbons in length, and containing a compound described herein or a pharmaceutically acceptable salt or prodrug thereof sufficient to preferentially deliver (i.e., target) an amount of the compound to the microvascular bed surrounding the alveoli in the lung.

The compounds described herein may be combined with other anti-influenza agents as also described herein. Such additional agents may also be present in the liposomes, may be present in different liposomes, or may be co-administered by different routes.

The liposomes include one or more of the compounds described herein or a pharmaceutically acceptable salt thereof, and may optionally include other anti-influenza agents. Liposomes can be prepared by dissolving phospholipids and cholesterol in a suitable organic solvent such as chloroform and evaporating the solvent to form a lipid film. If an ionophore is employed to load the compounds described herein into liposomes, the ionophore may be added to the lipid solution prior to evaporation. The dried lipid film is then rehydrated in a suitable aqueous phase, such as phosphate buffered saline or other physiologically suitable solution. The water soluble drug or therapeutic agent may be contained in the hydration solution, but if remote loading is desired, a loading agent such as the chelating agents described above may be added to the hydration solution to be encapsulated within the internal aqueous space of the liposome.

Upon addition of the hydration solution, liposomes of different sizes spontaneously form and encapsulate a portion of the aqueous phase. Thereafter, the liposomes and the aqueous suspension solution are subjected to a shearing force such as extrusion, sonic treatment or treatment by a homogenizer according to the method described in U.S. Pat. No. 4,753,788; to produce vesicles within a specified size.

The liposomes can then be treated to remove undesired compounds, such as unencapsulated drugs, from the suspension solution, which can be accomplished by processes such as gel chromatography or ultrafiltration.

The use of liposomes in dry powder aerosols for targeted Lung delivery is described, for example, in Willis et al, lungs (Lung), 6.2012, 190(3): 251-262.

Method of administration

Depending on the severity of the infection being treated, the compounds and pharmaceutically acceptable compositions described above may be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as powders, ointments, or drops), buccally, as an oral or nasal spray, to the pulmonary system (e.g., by using an inhaler, such as a Metered Dose Inhaler (MDI)), or the like.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art (e.g., water or other solvents), solubilizing agents and emulsifiers (e.g., ethyl alcohol, isopropyl alcohol, ethyl carbonate, EtOAc, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide), oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Injectable preparations (e.g., sterile injectable aqueous or oleaginous suspensions) can be formulated according to the known art using suitable dispersing, wetting and/or suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S. P., and isotonic sodium chloride solution. In addition, sterile, non-volatile oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland non-volatile oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids (e.g., oleic acid) are used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

To prolong the effect of the compounds described herein, it is generally desirable to slow the absorption of the compounds from subcutaneous or intramuscular injection. This can be achieved by using liquid suspensions of crystalline or amorphous materials which are poorly water soluble. The rate of absorption of the compound depends on its rate of dissolution, which may in turn depend on crystal size and crystalline form. Alternatively, delayed absorption of a compound administered parenterally is achieved by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming a microcapsule matrix of the compound in a biodegradable polymer, such as polylactide-polyglycolide. Depending on the ratio of compound to polymer and the nature of the particular polymer used, the rate of release of the compound can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Injectable depot formulations can also be prepared by encapsulating the compounds in liposomes or microemulsions that are compatible with body tissues.

Compositions for rectal or vaginal administration are in particular suppositories which can be prepared by mixing the compounds described herein with suitable non-irritating excipients or carriers, for example cocoa butter, polyethylene glycol or a suppository wax, which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, lozenges, pills, powders and granules. In such solid dosage forms, the active compound is mixed with at least one inert pharmaceutically acceptable excipient or carrier (e.g., sodium citrate or dicalcium phosphate) and/or: a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders such as carboxymethyl cellulose, alginate, gelatin, polyvinyl pyrrolidone, sucrose and acacia; c) humectants, such as glycerol; d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) solution retarding agents, such as paraffin; f) absorption accelerators, such as quaternary ammonium compounds; g) wetting agents, such as cetyl alcohol and glycerol monostearate; h) adsorbents such as kaolin and bentonite; and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof. In the case of capsules, lozenges, and pills, the dosage forms may also include buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft-filled and hard-filled gelatin capsules using excipients such as lactose (lactose/milk sugar) and high molecular weight polyethylene glycols and the like. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. It may optionally contain opacifying agents and may also have a composition that releases the active ingredient only or preferentially in a certain portion of the intestinal tract, or optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft-filled and hard-filled gelatin capsules using excipients such as lactose (lactose/milk sugar) and high molecular weight polyethylene glycols and the like.

The active compound may also be in microencapsulated form with one or more excipients as mentioned above. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shell layers such as enteric coatings, release controlling coatings, and other coatings well known in the pharmaceutical formulating art. In these solid dosage forms, the active compound may be mixed with at least one inert diluent (e.g., sucrose, lactose or starch). As in general practice, such dosage forms may also include other substances in addition to inert diluents, such as tableting lubricants and other tableting aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, lozenges, and pills, the dosage forms may also comprise buffering agents. It may optionally contain opacifying agents and may also have a composition that releases the active ingredient only or preferentially in a certain portion of the intestinal tract, or optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of the compounds described herein include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active ingredient is admixed under sterile conditions with a pharmaceutically acceptable carrier and any required preservatives or buffers as may be desired. Also contemplated are ophthalmic formulations, ear drops, and eye drops that are within the scope of the present disclosure. In addition, the present disclosure contemplates the use of transdermal patches, which have the additional advantage of providing controlled delivery of compounds to the body. Such dosage forms may be prepared by dissolving or dispensing the compound in the appropriate medium. Absorption enhancers may also be used to increase the flux of the compound through the skin. The rate can be controlled by providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

The compositions described herein may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implantable reservoir. The term "parenteral" as used herein includes, but is not limited to, subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. In particular, the compositions are administered orally, intraperitoneally, or intravenously.

The sterile injectable form of the compositions described herein can be an aqueous or oleaginous suspension. These suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, in solution in 1, 3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. In addition, sterile, non-volatile oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland non-volatile oil may be employed including synthetic mono-or diglycerides. Fatty acids (e.g., oleic acid and its glyceride derivatives) are suitable for use in the preparation of injectables, as are natural pharmaceutically-acceptable oils (e.g., olive oil or castor oil, especially in their polyoxyethylated versions). These oil solutions or suspensions may also contain a long chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersants commonly used in formulating pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants (e.g., Tween, Span, and other emulsifiers) or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for formulation purposes.

The pharmaceutical compositions described herein may be administered orally in any orally acceptable dosage form including, but not limited to, capsules, lozenges, aqueous suspensions or solutions. In the case of lozenges for oral use, common carriers include, but are not limited to, lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in capsule form, suitable diluents include lactose and dried corn starch. When aqueous suspensions are desired for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.

Alternatively, the pharmaceutical compositions described herein may be administered in the form of suppositories for rectal administration. These suppositories can be prepared by mixing the agent with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions described herein may also be administered topically, particularly when the target of treatment includes topical application of readily accessible areas or organs, including diseases of the eye, skin or lower intestinal tract. Suitable topical formulations for each of these regions or organs are readily prepared.

Topical administration for the lower intestinal tract may be achieved in the form of a rectal suppository formulation (see above) or in a suitable enema formulation. Topical transdermal patches may also be used.

For topical administration, the pharmaceutical compositions may be formulated in a suitable ointment containing the active ingredient suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds described herein include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyethylene oxide, polypropylene oxide compounds, emulsifying waxes, and water. Alternatively, the pharmaceutical compositions may be formulated in a suitable lotion or cream containing the active ingredient suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

For ocular use, the pharmaceutical composition may be formulated as a micronized suspension in isotonic, pH adjusted, sterile saline, or specifically as a solution in isotonic, pH adjusted, sterile saline, with or without a preservative (e.g., benzalkonium chloride). Alternatively, for ophthalmic use, the pharmaceutical composition may be formulated in an ointment such as petrolatum.

The compounds for use in the methods described herein may be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for the individual undergoing treatment, wherein each unit contains a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form may be employed in a single daily dose or in one of a plurality of daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form for each dose may be the same or different.

The present disclosure may be more completely understood in consideration of the examples described herein in detail in connection with the detailed description of exemplary embodiments. However, these examples should not be construed as limiting the scope of the disclosure. All references throughout this disclosure are expressly incorporated by reference herein.

Examples of the invention

Example 1 cytopathic Effect analysis

A study was conducted to evaluate the in vitro combined effect of the test compound 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid with other influenza antiviral agents in an influenza cell-based cytopathic effect (CPE) assay.

Influenza A/PR/8/34 virus (H1N1) (ATCC VR-1469) was obtained from ATCC. MDCK cells (ATCC CCL-34) were obtained from ATCC and maintained in minimal essential eagle medium (Sigma M2279) supplemented with 10% fetal bovine serum (Corning R35-076-CV), 1% L-glutamic acid (Gibco 25030081), 1% non-essential amino acids (NEAA, Gibco 11140050) and 1% penicillin-streptomycin (PS, Hyclone SV 30010).

The test medium for MDCK cells was OptiPRO SFM medium (Gibco 12309019) supplemented with 1% L-glutamic acid, 1% NEAA, 1% PS, and 2.5 μ g/mL trypsin was used as the test medium for MDCK cells.

In 384-well plates, MDCK cells were seeded at 2,000 cells/well and at 37 ℃ and 5% CO₂Incubate overnight. The following day, each of the 2 drug combination pairs was tested using a checkerboard-like cross pattern of seven drug concentrations for each compound, including triplicate plates of each compound alone. Pimodivir (VX-787) was evaluated in parallel as a control compound. Compounds (in DMSO) were inoculated by Tecan HP D300 digital dispenser. Followed by addition of Virus (2 TCID)₉₀Hole/bore). The tested concentrations of the compounds were 0.125, 0.25, 0.5, 1,2, 4 and 8 × EC₅₀The value is obtained. The compound combinations are listed in table 4. The final concentration of DMSO (Sigma34869) in cell culture medium was 0.5%. The resulting culture was incubated at 37 ℃ and 5% CO₂Incubations were continued for 5 additional days until viral infection in the virus control showed significant CPE (as assessed by a decrease in viable cell number). To assess cell viability, CCK-8 kit solution (a vial of colorimetric system, Biolite 35004) was then added to each well and the cells were incubated at 37 ℃ for 3 hours. Absorbance (460nm) was measured using a SpectraMax 340PC384 disc reader (molecular device).

The antiviral activity and cytotoxicity of the compound combinations were expressed as% inhibition and% survival, respectively, and were calculated by the following equations:

inhibition (%) (raw data)_cpd-average_VC) /(average)_CC-average_VC)×100

Survival (%) (raw data)_cpd-average_MC) /(average)_CC-average_MC)×100

Raw data_cpdRepresenting the absorbance value of the compound-treated wells; average_VCAverage, average_CCAnd average_MCMean absorbance values for virus control (VC; virus-infected cells, no compound), cell control (CC; cells without virus or compound), and medium control (MC, medium only), respectively. Using MacSynergy^TMII software (Prichard and Shipman, 1990) to calculate the combination index. Subsequent computing of synergiesThe dosage graph (95%). Positive combination index values indicate synergy and negative combination index values indicate antagonism.

As shown in the table below, 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid demonstrates a strong synergistic effect with influenza antiviral drugs, including baroxavir (without the hydroxy form of the baroxavir macbecate prodrug), oseltamivir acid, and favipiravir, in an influenza antiviral CPE assay against IFV a/PR/8/34(H1N 1).

An absolute combination index value <25 (i.e., 95% synergy) represents an additive effect. Absolute combination index values 25 to 50 indicate slight synergy or antagonism. Absolute combination index values of 50 to 100 indicate moderate synergy or antagonism. Absolute combination index value >100 indicates a strong synergy or antagonism.

Fig. 1-3 provide visual illustrations of respective calculation plots for three test combinations.

These same three combinations of compound 1 with baloxavir (without the hydroxy form of the baloxavir boswellate prodrug), oseltamivir acid and favipiravir also exhibited antiviral activity against influenza B.

Claims (62)

1. A method of treating or preventing influenza virus infection or replication in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of (1)3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid or a pharmaceutically acceptable salt or solvate thereof, and (2) a second antiviral agent.

2. The method of claim 1, wherein the second antiviral agent is a neuraminidase inhibitor, a polymerase inhibitor, an endonuclease inhibitor or an influenza vaccine.

3. The method of claim 1, wherein the second antiviral agent is selected from the group consisting of: baculosaprolimus, baculosapivir, neuraminidase inhibitors and favipiravir, or pharmaceutically acceptable salts or solvates thereof.

4. The method of claim 2, wherein the neuraminidase inhibitor is oseltamivir, oseltamivir acid, zanamivir, lanamivir, peramivir, or a pharmaceutically acceptable salt or solvate thereof.

5. The method of claim 4, wherein the neuraminidase inhibitor is oseltamivir, oseltamivir acid, or a pharmaceutically acceptable salt or solvate thereof.

6. The method of claim 1, wherein the second antiviral agent is baroxavir bociclate, baroxavir, or a pharmaceutically acceptable salt or solvate thereof.

7. The method of claim 1, wherein the second antiviral agent is fapiravir or a pharmaceutically acceptable salt or solvate thereof.

8. The method of claim 1, wherein the second anti-viral agent is an influenza vaccine.

9. The method of any one of claims 1 to 8, wherein the 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid or a pharmaceutically acceptable salt or solvate thereof is administered prior to the second antiviral agent.

10. The method of any one of claims 1 to 8, wherein the 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid or a pharmaceutically acceptable salt or solvate thereof is administered after the second anti-viral agent.

11. The method of any one of claims 1 to 8, wherein the 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid, or a pharmaceutically acceptable salt or solvate thereof, is administered concurrently with the second antiviral agent.

12. The method of claim 11, wherein the 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid, or a pharmaceutically acceptable salt or solvate thereof, is co-formulated with the second antiviral agent.

13. The method of claim 11, wherein the 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid, or a pharmaceutically acceptable salt or solvate thereof, and the second antiviral agent are in separate formulations.

14. A combination comprising 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid or a pharmaceutically acceptable salt or solvate thereof and a second antiviral agent.

15. The combination of claim 14, wherein the second antiviral agent is a neuraminidase inhibitor, a polymerase inhibitor, an endonuclease inhibitor or an influenza vaccine.

16. The combination of claim 14, wherein the second antiviral agent is selected from the group consisting of: barosavirenz, Barosavir, neuraminidase inhibitors and Favipiravir, as well as pharmaceutically acceptable salts or solvates thereof.

17. A combination comprising a) a therapeutically effective amount of 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid or a pharmaceutically acceptable salt or solvate thereof and b) a therapeutically effective amount of a second antiviral agent.

18. The combination of claim 17, wherein the second antiviral agent is a neuraminidase inhibitor, a polymerase inhibitor, an endonuclease inhibitor or an influenza vaccine.

19. The combination of claim 17, wherein the second antiviral agent is selected from the group consisting of: barosavir, Barosavira bociclate, a neuraminidase inhibitor, and Favipiravir or a pharmaceutically acceptable salt or solvate thereof.

20. The combination according to claims 11-19, wherein the neuraminidase inhibitor is oseltamivir, oseltamivir acid, zanamivir, lanamivir, peramivir, or a pharmaceutically acceptable salt or solvate thereof.

21. The combination according to claim 20, wherein the neuraminidase inhibitor is oseltamivir, oseltamivir acid or a pharmaceutically acceptable salt or solvate thereof.

22. The combination of claim 14 or 17, wherein the second antiviral agent is baroxavir bociclylate, baroxavir or a pharmaceutically acceptable salt or solvate thereof.

23. The combination according to claim 14 or 17, wherein the second antiviral agent is fapiravir or a pharmaceutically acceptable salt or solvate thereof.

24. The combination of claim 14 or 17, wherein the second anti-viral agent is an influenza vaccine.

25. Use of a therapeutically effective amount of a combination according to any one of claims 14 to 24 for the treatment or prevention of influenza virus infection or replication in a human patient.

26. The use of claim 25, wherein the influenza virus is a pandemic or drug resistant pandemic. Sexual/seasonal influenza virus.

27. Use of 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid, or a pharmaceutically acceptable salt or solvate thereof, in combination with a second antiviral agent in the manufacture of a medicament for the treatment or prevention of an influenza virus infection or replication.

28. Use of 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid, or a pharmaceutically acceptable salt or solvate thereof, in combination with a second antiviral agent, for inhibiting influenza virus infection or replication.

29. The use of claim 27 or 28, wherein the second antiviral agent is a neuraminidase inhibitor, a polymerase inhibitor, an endonuclease inhibitor or an influenza vaccine.

30. The use of claim 27 or 28, wherein the second antiviral agent is selected from the group consisting of: barosavir, Barosavira bociclate, a neuraminidase inhibitor, and Favipiravir or a pharmaceutically acceptable salt or solvate thereof.

31. The use of claim 27 or 28, wherein the second anti-viral agent is an influenza vaccine.

32. The use of claim 27 or 28, wherein the second antiviral agent is a neuraminidase inhibitor selected from: oseltamivir, oseltamivir acid, zanamivir, lanamivir, peramivir or a pharmaceutically acceptable salt or solvate thereof.

33. The use of claim 32, wherein the neuraminidase inhibitor is oseltamivir, oseltamivir acid or a pharmaceutically acceptable salt or solvate thereof.

34. The use of claim 27 or 28, wherein the second antiviral agent is baroxavir bociclylate, baroxavir or a pharmaceutically acceptable salt or solvate thereof.

35. The use of claim 27 or 28, wherein the second antiviral agent is fapiravir or a pharmaceutically acceptable salt or solvate thereof.

36. The use of claim 27 or 28, wherein the second anti-viral agent is an influenza vaccine.

37. A method for treating or preventing influenza virus infection or replication, the method comprising administering to a human patient having or at risk of influenza virus infection a combination of: a) a therapeutically effective amount of 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid or a pharmaceutically acceptable salt or solvate thereof; and b) a therapeutically effective amount of a second anti-viral agent.

38. The method of claim 37, wherein the second antiviral agent is a neuraminidase inhibitor, a polymerase inhibitor, an endonuclease inhibitor or an influenza vaccine.

39. The method of claim 37, wherein the second anti-viral agent is selected from the group consisting of: barosavir, Barosavira bociclate, a neuraminidase inhibitor, and Favipiravir or a pharmaceutically acceptable salt or solvate thereof.

40. A method for treating or preventing influenza virus infection or replication, the method comprising administering to a human patient having or at risk of influenza infection a dose of about 10 to 1,000mg/kg of 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid or a pharmaceutically acceptable salt or solvate thereof and a therapeutically effective amount of a second antiviral agent.

41. The method of claim 40, wherein the second antiviral agent is a neuraminidase inhibitor, a polymerase inhibitor, an endonuclease inhibitor or an influenza vaccine.

42. The method of claim 40, wherein the second anti-viral agent is selected from the group consisting of: barosavir, Barosavira bociclate, a neuraminidase inhibitor, and Favipiravir or a pharmaceutically acceptable salt or solvate thereof.

43. The method of claim 39 or 42, wherein the neuraminidase inhibitor is oseltamivir, oseltamivir acid, zanamivir, lanamivir, peramivir, or a pharmaceutically acceptable salt or solvate thereof.

44. The method of claim 43, wherein the neuraminidase inhibitor is oseltamivir, oseltamivir acid, or a pharmaceutically acceptable salt or solvate thereof.

45. The method of claim 39 or 42, wherein the second antiviral agent is Barosavirenz bociclate, Barosavir, or a pharmaceutically acceptable salt or solvate thereof.

46. The method of claim 39 or 42, wherein the second antiviral agent is Favipiravir or a pharmaceutically acceptable salt or solvate thereof.

47. The method of claim 37 or 40, wherein the second anti-viral agent is an influenza vaccine.

48. A method of inhibiting the endonuclease activity of an influenza polymerase in an influenza a or B virus, the method comprising contacting the virus with the combination of any one of claims 11 to 16.

49. A method for the treatment or prophylaxis of influenza a or influenza B infection in a host, said method comprising administering to said host a therapeutic amount of a combination according to any one of claims 11 to 16.

50. A method for reducing the endonuclease activity of an influenza a polymerase or an influenza B polymerase in a host, said method comprising administering to said host a therapeutic amount of a combination according to any one of claims 11 to 16.

51. A method for reducing influenza virus replication in a host, the method comprising administering to the host a therapeutic amount of a combination according to any one of claims 14 to 24.

52. The method of any one of claims 37-51, further comprising contacting the influenza virus with or administering to the host a therapeutically effective amount of a third antiviral agent.

53. The method of claim 51 or 52, further comprising administering an influenza vaccine to the host before, after, or simultaneously with the combining.

54. The method of any one of claims 37-53, wherein the influenza virus is a pandemic or drug resistant pandemic/seasonal influenza virus.

55. Use of a combination according to any one of claims 14 to 24 for the treatment of influenza a or influenza B virus infection.

56. Use of a combination according to any one of claims 14 to 24 in the manufacture of a medicament for the treatment of influenza a or influenza B virus infection.

57. A pharmaceutical composition comprising 3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid, or a pharmaceutically acceptable salt or solvate thereof, for use in treating or preventing an influenza virus infection or replication in a patient, wherein the composition is administered in combination with a second antiviral agent.

58. The composition of claim 57, wherein the second antiviral agent is a neuraminidase inhibitor, a polymerase inhibitor, an endonuclease inhibitor or an influenza vaccine.

59. The composition of claim 57, wherein the second antiviral agent is selected from the group consisting of: barosavir, Barosavira bociclate, a neuraminidase inhibitor, and Favipiravir or a pharmaceutically acceptable salt or solvate thereof.

60. A combination comprising a)3- (2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-7H-pyrrolo [2,3-d ] pyrimidin-7-yl) bicyclo [2.2.2] octane-2-carboxylic acid or a pharmaceutically acceptable salt or solvate thereof, b) baroxavir, or a pharmaceutically acceptable salt or solvate thereof, and c) a neuraminidase inhibitor.

61. The composition or combination of any one of claims 57-60, wherein the neuraminidase inhibitor is oseltamivir, oseltamivir acid, zanamivir, lanamivir, peramivir, or a pharmaceutically acceptable salt or solvate thereof.

62. The composition or combination of claim 61, wherein the neuraminidase inhibitor is oseltamivir, oseltamivir acid, or a pharmaceutically acceptable salt or solvate thereof.

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